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A structure-activity relationship study of dorsomorphin, a previously identified inhibitor of SMAD 1/5/8 phosphorylation by bone morphogenetic protein (BMP) type 1 receptors ALK2, 3, and 6, revealed that increased inhibitory activity could be accomplished by replacing the pendent 4-pyridine ring with 4-quinoline. The activity contributions of various nitrogen atoms in the core pyrazolo[1,5-a]pyrimidine ring were also examined by preparing and evaluating pyrrolo[1,2-a]pyrimidine and pyrazolo[1,5-a]pyridine derivatives. In addition, increased mouse liver microsome stability was achieved by replacing the ether substituent on the pendent phenyl ring with piperazine. Finally, an optimized compound 13 (LDN-193189 or DM-3189) demonstrated moderate pharmacokinetic characteristics (e.g., plasma t(1/2)=1.6h) following intraperitoneal administration in mice. These studies provide useful molecular probes for examining the in vivo pharmacology of BMP signaling inhibition.

Cyclin-dependent kinase 5 (cdk5) is a member of the serine-threonine kinase family of cyclin-dependent kinases. This family is known for its role in the cell cycle, but cdk5 differs due to its interaction with activators p35 or p39, both abundant in post-mitotic neurons. Cdk5 is not known to have a role in cell cycle regulation at all, but is known to be an important modulator of neuronal activity. Cdk5 has been an attractive target for CNS diseases for a number of years. Among its attractions is the possibility that inhibitors will prevent the pathological phosphorylation of tau and neurofibrillary pathology in both Alzheimer's disease and tauopathies. More recently, there has been evidence that cdk5 is involved in the processing of pain and therefore inhibitors would also have potential therapeutic value for acute pain. Several classes of potent chemical inhibitors for cdk5 have been identified but most are competitive with the ATP binding site, resulting in a lack of specificity among the other cyclin-dependent kinases as well as other ATP-dependent kinases. We are working to discover specific inhibitors that might disrupt the interaction of tau and cdk5 at sites other than the ATP binding site. We are screening our compound library of 110,000 compounds using the full length tau as a substrate and will separate ATP competitive from non-competitive binders. In addition, we are taking a computational approach with virtual screening to identify non-ATP-competitive binders. These two approaches may lead to the discovery of site-specific inhibitors for tau and cdk5 interactions rather than competitive inhibitors for ATP binding. The hope is that non-ATP competitive compounds will more likely be selective and will be better therapeutics.

Necroptosis is a cellular mechanism of necrotic cell death induced by apoptotic stimuli in the form of death domain receptor engagement by their respective ligands under conditions where apoptotic execution is prevented. Although it occurs under regulated conditions, necroptotic cell death is characterized by the same morphological features as unregulated necrotic death. Here we report that necrostatin-1, a previously identified small-molecule inhibitor of necroptosis, is a selective allosteric inhibitor of the death domain receptor-associated adaptor kinase RIP1 in vitro. We show that RIP1 is the primary cellular target responsible for the antinecroptosis activity of necrostatin-1. In addition, we show that two other necrostatins, necrostatin-3 and necrostatin-5, also target the RIP1 kinase step in the necroptosis pathway, but through mechanisms distinct from that of necrostatin-1. Overall, our data establish necrostatins as the first-in-class inhibitors of RIP1 kinase, the key upstream kinase involved in the activation of necroptosis.

Necroptosis is a regulated caspase-independent cell death mechanism that can be induced in multiple cell types and is characterized by morphological features resembling necrosis. Here we describe a series of tricyclic heterocycles (i.e., 3-phenyl-3,3a,4,5-tetrahydro-2H-benz[g]indazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzopyrano[4,3-c]pyrazoles, 3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles, and 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro[1]benzothiopyrano[4,3-c]pyrazoles], collectively termed Nec-3, that can potently inhibit necroptosis. For example, compounds 8, 22, 41, 53, and 55 inhibit necroptosis in an FADD-deficient variant of human Jurkat T cells treated for 24 h with TNF-alpha with EC50 values in the range 0.15-0.29 microM. Distinct from the previously described series of hydantoin-containing indole derivatives (Nec-1), the Nec-3 series exhibits specificity in inhibiting TNF-alpha-induced necroptosis. A structure-activity relationship (SAR) study revealed that the (3R,3aR)-rel-diastereomers were more active than the (3R,3aS)-rel-diastereomers for all four ring systems. Introduction of fluorine or methoxy to the 8-position of the tricyclic ring and a methoxy to the 4-position of the pendent phenyl ring increased activity. Amides at the 2-position of the tricyclic ring were best. The Nec-3 series provides new tools for elucidating caspase-independent cell death pathways and potentially lead compounds for therapeutic development.

3-Amino-2-keto-7H-thieno[2,3-b]pyridin-6-one derivatives were discovered as moderately potent inhibitors of ubiquitin C-terminal hydrolase-L1 (UCH-L1) utilizing an assay that measures hydrolysis of the fluorogenic substrate Ub-AMC. SAR studies revealed that both the carboxylate at the 5-position and the 6-pyridone ring were critical for inhibitory activity. Furthermore, activity was dependent on the nature of the ketone substituent at the 2-position, with 4-Me-Ph and 2-naphthyl being best. Kinetic mechanism studies revealed that these compounds were uncompetitive inhibitors of UCH-L1, binding only to the Michaelis-complex and not to free enzyme. The active compounds were selective for UCH-L1, exhibiting neither inhibition of other cysteine hydrolases (e.g., UCH-L3, papain, isopeptidase T, caspase-3, and tissue transglutaminase) nor cytotoxicity in N2A cells.

The photostimulated intramolecular ortho-arylation reactions of bromoarenes linked with pendant phenoxy containing N-substituted tetrahydroisoquinolines in liquid ammonia afforded aporphine (54−82% yield) alkaloid derivatives via SRN1 reactions. This strategy was extended for the first time to the synthesis of a homoaporphine derivative (40% yield). Tetrahydroisoquinoline precursors that contained electron-withdrawing groups on nitrogen (i.e., amides, sulfonamides, and carbamates) gave cyclized products, whereas precursors with basic nitrogens (i.e., NH or NMe) either failed to yield cyclized products or gave aporphines in only low yield.

Tuberculosis (TB) remains a worldwide problem and the need for new drugs is increasingly more urgent with the emergence of multidrug- and extensively-drug resistant TB. Inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis (Mtb) is an attractive drug target. The enzyme catalyzes the conversion of inosine 5'-monophosphate into xanthosine 5'-monophosphate with the concomitant reduction of NAD+ to NADH. This reaction controls flux into the guanine nucleotide pool. We report seventeen selective IMPDH inhibitors with antitubercular activity. The crystal structures of a deletion mutant of MtbIMPDH2 in the apo form and in complex with the product XMP and substrate NAD+ are determined. We also report the structures of complexes with IMP and three structurally distinct inhibitors, including two with antitubercular activity. These structures will greatly facilitate the development of MtbIMPDH2-targeted antibiotics.

The photostimulated intramolecular ortho-arylation reactions of bromoarenes linked with pendant phenoxy containing N-substituted tetrahydroisoquinolines in liquid ammonia afforded aporphine (54-82% yield) alkaloid derivatives via SRN1 reactions. This strategy was extended for the first time to the synthesis of a homoaporphine derivative (40% yield). Tetrahydroisoquinoline precursors that contained electron-withdrawing groups on nitrogen (i.e., amides, sulfonamides, and carbamates) gave cyclized products, whereas precursors with basic nitrogens (i.e., NH or NMe) either failed to yield cyclized products or gave aporphines in only low yield.

Six sets of teneral Glossina palpalis gambiensis (Diptera: Glossinidae) were fed on mice infected with six different isolates of Trypanosoma brucei gambiense (each mouse was infected with one of the isolates), previously isolated from patients in the sleeping sickness focus of Bonon, Cote d’Ivoire and in Makoua, Congo. All the tsetse flies were dissected 42 days post-infection and midgut and salivary glands were examined for trypanosomes by microscopical examination. No infection was observed with the reference stock whereas each of the five recently isolated trypanosome isolates was able to infect tsetse flies, with rates of infection varying between 9.7 and 18.2% depending on the isolate. Three isolates displayed only immature infections with 9.7, 17.3 and 18% of the flies showing trypanosomes in their midgut. One isolate gave both immature (12.1%) and mature infections (6.1%). Finally, the last isolate involved only mature infections in 9.7% of the Glossina species examined. These substantial differences in the cyclical transmission of T. b. gambiense in the same fly species could have important implications for the epidemiology of the transmission of Human African Trypanosomiasis.

Thieno[2,3-d]pyrimidin-4-one acylhydrazide derivatives were discovered as moderately potent inhibitors of TGase 2 (tissue transglutaminase) utilizing a fluorescence-based assay that measured TGase 2 catalyzed incorporation of the dansylated Lys derivative alpha-N-Boc-Lys-CH(2)-CH(2)-dansyl into the protein substrate N,N-dimethylated-casein. A SAR study revealed that the acylhydrazide thioether side-chain and the thiophene ring were critical to inhibitory activity.

Necroptosis is a regulated caspase-independent cell death mechanism that results in morphological features resembling necrosis. It can be induced in a FADD-deficient variant of human Jurkat T cells treated with TNF-alpha. 5-(1H-Indol-3-ylmethyl)-2-thiohydantoins and 5-(1H-indol-3-ylmethyl)hydantoins were found to be potent necroptosis inhibitors (called necrostatins). A SAR study revealed that several positions of the indole were intolerant of substitution, while small substituents at the 7-position resulted in increased inhibitory activity. The hydantoin ring was also quite sensitive to structural modifications. A representative member of this compound class demonstrated moderate pharmacokinetic characteristics and readily entered the central nervous system upon intravenous administration.

SummaryCyclin-dependent kinase 5 (Cdk5) is widely viewed as a possible target for a wide variety of neurological disorders. One pathological role attributed to Cdk5 is the abnormal phosphorylation of tau that may lead to the neuronal inclusions known as neurofibrillary tangles. A high through-put screen for inhibitors of Cdk5-mediated phosphorylation of tau resulted in three compounds with distinct mechanisms of action. One compound is competitive with ATP and has a high affinity for the Cdk5 ATP binding pocket. The second compound also competes with ATP, is noncompetitive with tau, and (uniquely among this class of inhibitors) displaces adjacent amino acid residues to make room for the nitrophenyl group. A third compound did not compete with ATP, but did compete with tau at low concentrations of tau. The SAR and charge optimization derived from cocrystals of the two ATP competitors along with cocrystals of three other ATP competitors map out the importance of filling and properly charging different regions of the ATP binding pocket. Taken together, this analysis shows how the structure of Cdk5 constrains the space of potential inhibitors and reveals a pocket unfilled in all of the structures. These leads could be a starting point for structure-based drug design of more potent and selective inhibitors.

The mechanism of apoptosis has been extensively characterized over the past decade, but little is known about alternative forms of regulated cell death. Although stimulation of the Fas/TNFR receptor family triggers a canonical 'extrinsic' apoptosis pathway, we demonstrated that in the absence of intracellular apoptotic signaling it is capable of activating a common nonapoptotic death pathway, which we term necroptosis. We showed that necroptosis is characterized by necrotic cell death morphology and activation of autophagy. We identified a specific and potent small-molecule inhibitor of necroptosis, necrostatin-1, which blocks a critical step in necroptosis. We demonstrated that necroptosis contributes to delayed mouse ischemic brain injury in vivo through a mechanism distinct from that of apoptosis and offers a new therapeutic target for stroke with an extended window for neuroprotection. Our study identifies a previously undescribed basic cell-death pathway with potentially broad relevance to human pathologies.

A new route to substituted β -carbolines and carbazoles is described. Diketoindole intermediates, prepared by Friedel–Crafts acylations of 3-substituted indoles, have been converted to 3-hydroxycarbazoles and β -carbolines in good yields, 51–96% and 82–97%, respectively. This method also allows for the formation of 4-substituted β -carbolines. The application of this methodology to the synthesis of the natural products hyellazole and 6-chlorohyellazole is also described.

A synthesis of the alkaloid (±)-aporphine is reported. The initial key step of the synthesis involves a Pictet–Spengler cyclization of N-tosyl tyramine with 2-bromophenylacetaldehyde in trifluoroacetic acid. This step was followed by the second strategic transformation a palladium-mediated intramolecular phenol ortho -arylation reaction utilizing tricyclohexylphosphine as co-catalysts in the presence of cesium carbonate. Finally, de-oxygenation of the phenol, removal of the tosyl group and methylation gave the desired alkaloid.

The 5' untranslated region (5'UTR) of the transcript encoding the Alzheimer's amyloid precursor protein (APP) is a key regulatory sequence that determines the amount of intracellular APP holoprotein present in brain derived cells. Using neuroblastoma cells (SY5Y) we developed a transfection based screen of a library of FDA drugs to identify compounds that limited APP luciferase reporter expression translated from the APP 5'UTR. Paroxetine (Paxil trade mark ), dimercaptopropanol, phenserine, desferrioxamine, tetrathiolmobdylate, and azithromycin were six leads that were subsequently found to also suppress APP holoprotein levels or to alter APP cleavage (azithromycin). Since APP holoprotein levels are proportionate to Abeta peptide output in many systems we tested the efficacy of paroxetine and dimercaptopropanol to limit Abeta secretion as measured by ELISA assays. Paroxetine and dimercaptopropanol limited Abeta peptide secretion from lens epithelial cells (B3 cells). Interestingly, paroxetine changed the steady-state levels of transferrin receptor mRNAs. These data suggested that this serotonin reuptake inhibitor (SSRI) provided extra pharmacological action to chelate interacellular iron or change the intracellular iron distribution. An altered iron distribution would be predicted to indirectly limit APP holoprotein expression and Abeta peptide secretion.

The four stereoisomers of mesoridazine were synthesized and evaluated in D2, 5-HT1A, 5-HT2A, 5-HT2C, D1, and D3 receptor binding and functional assays. Two isomers demonstrated potent D2 receptor binding (Ki3 nM) and functional antagonism (IC50or = 10 nM) activities. These two isomers also showed moderate affinity for the 5-HT2A and D3 receptors. A third isomer was devoid of significant D2 receptor binding, but did have moderate affinity for the 5-HT2A and D3 receptors. The fourth isomer demonstrated poor affinity for all the receptors tested. Most significantly, the stereochemistry of the sulfoxide moiety played a dominant role in the observed structure-activity relationship (SAR).

A palladium-mediated intramolecular phenol ortho -arylation reaction applied to the construction of aporphine alkaloids is reported. Most significantly, the efficiency of this transformation was enhanced by the utilization of trialkylphosphine (i.e. tricyclohexylphosphine) or trialkylphosphonium salts (i.e. di- tert -butylmethyl-phosphonium tetrafluoroborate) as co-catalysts in the presence of cesium carbonate. This methodology was employed in the syntheses of the aporphine alkaloids (±)-lirinidine and (±)-nuciferine.

Cryptosporidium parvum is a potential biowarfare agent, an important AIDS pathogen, and a major cause of diarrhea and malnutrition. No vaccines or effective drug treatment exist to combat Cryptosporidium infection. This parasite relies on inosine 5'-monophosphate dehydrogenase (IMPDH) to obtain guanine nucleotides, and inhibition of this enzyme blocks parasite proliferation. Here, we report the first crystal structures of CpIMPDH. These structures reveal the structural basis of inhibitor selectivity and suggest a strategy for further optimization. Using this information, we have synthesized low-nanomolar inhibitors that display 10(3) selectivity for the parasite enzyme over human IMPDH2.

The potent photoactivated virucidal activity of tridentate 2,2'-dihydroxyazobenzene- and 2-salicylideneaminophenol platinum pyridine complexes 1, 2, 4, 6, 7, 9, and 10 against enveloped viruses (e.g., EIAV, HIV, and HSV) is described.

ID1 (inhibitor of DNA-binding-1) is a member of the helix-loop-helix family of transcriptional regulatory proteins. The ID-family of proteins (ID1-ID4) inhibit the DNA binding of transcription factors which regulate cellular differentiation and proliferation. Accordingly, deregulation of ID proteins has been observed in many cancer types including leukemia. High levels of ID1 expression are found in primary acute myeloid leukemia (AML) samples and correlate with poor prognosis. ID1 is also identified as a common downstream target of the oncogenic tyrosine kinases, BCR-ABL, TEL-ABL and FLT3-ITD. In addition, Id1 has been shown to promote a myeloproliferative disease in mice, and knockdown of ID1 expression inhibits leukemic cell growth. Therefore, ID1 is an excellent candidate for targeted therapy in leukemia. However, suitable drugs to target ID1 have not been developed to date. ID1 is normally polyubiquitinated and degraded by the proteasome. Recently, it has been shown that USP1, a ubiquitin specific protease, deubiquitinates ID1 and rescues it from proteasome degradation. Inhibition of USP1 therefore offers a new avenue to target ID1 in cancer. Here, using a Ubiquitin-Rhodamine-based high throughput screen, we identified small molecule inhibitors of USP1 and investigated their therapeutic potential for leukemia. These inhibitors blocked the deubiquitinating enzyme activity of USP1 in vitro in a dose-dependent manner with an IC50 in the nanomolar range, and also targeted the enzyme activity of native USP1. To determine the cellular consequences of USP1 inhibition, we exposed leukemic cells to micromolar concentrations of the inhibitors and evaluated ID1 levels and survival. USP1 inhibitors promoted the degradation of ID1 and, concurrently, inhibited the growth (>90% inhibition in 24 hrs) of chronic myelogenous leukemia (CML) and AML cell lines with induction of apoptosis in a dose dependent manner. The EC50 of the inhibitors for the leukemic cell growth inhibition was approximately 1.07 μM ± 0.08 (95% Confidence Limits). Interestingly, exposure to low doses of USP1 inhibitor for 5 days in culture resulted in erythroid differentiation of K562 leukemic cells. A known USP1 inhibitor, Pimozide, also promoted ID1 degradation and inhibited growth of leukemic cells (>90% inhibition in 48 hrs), though at a higher drug concentrations as compared to the novel USP1 inhibitors. Importantly, the novel USP1 inhibitors promoted ID1 degradation and exhibited cytotoxicity (>90% death in 48 hrs) in primary AML patient-derived leukemic cells. Notably, siRNA-mediated knockdown of USP1 in K562 leukemic cells resulted in growth inhibition, increased apoptosis and cell cycle arrest. Collectively, our results demonstrate that the novel small molecule inhibitors of USP1 promote ID1 degradation and are cytotoxic to leukemic cells. The identification of USP1 inhibitors therefore opens up a new approach for leukemia therapy. Disclosures: No relevant conflicts of interest to declare.

Receptor Interacting Protein 1 (RIP1) kinase, a component of the Tumor Necrosis Factor alpha (TNFα) receptor signaling complexes, has recently emerged as a new important mediator of cell death signaling. Firstly, RIP1, in complex with a homologous RIP3 kinase, mediates activation of regulated necrotic death, termed necroptosis. Necroptosis is activated by TNFα family members as well as other signals under apoptosis-deficient conditions. Secondly, recent evidence suggests that in some cases RIP1 kinase can also contribute to the induction of apoptosis through the formation of the “ripoptosome” complex, leading to the activation of caspase-8. Ser/Thr kinase activity of RIP1 plays a critical role in the activation of both forms of cell death. Necrosis is a major form of cell death contributing to most acute pathologic injuries. Necroptosis displays all the major hallmarks of pathologic necrosis. This raises an intriguing possibility that RIP1-dependent regulated necroptosis, rather than the unregulated passive necrosis, may be responsible for the pathologic injury in vivo in various necrotizing diseases and, furthermore, that these injuries may be targeted therapeutically by inhibitors of necroptotic signaling. Indeed, emerging evidence suggests that inhibition of RIP1 kinase activity or genetic deletion of RIP3 kinase provides significant tissue and functional protection in a variety of mouse models of human disease, including those of stroke, myocardial infarction, retinal injury, septic shock and acute pancreatitis. On the other hand, recent evidence also suggest that under physiologic conditions necroptosis is activated as a secondary response to the genetic loss of several apical apoptosis regulators, such as caspase-8 and FADD, indicating that normally necroptosis may act as a surveillance mechanism ensuring the integrity of the apoptotic signaling machinery. In order to explore therapeutic potential of necroptosis inhibition, we identified several structurally diverse small molecule inhibitors of necroptosis, termed necrostatins, in a high throughput screen for the suppressors of TNFα-induced necroptotic death. These molecules are specific and efficient inhibitors of necrotic death in a variety of cellular models of necroptosis. Medicinal chemistry optimization of necrostatins led to significantly improved biologic activity, resulting in a panel of four diverse sub-micromolar, metabolically stable small molecule inhibitors of cell death. Our further studies indicated that even though necrostatins were selected in a random cell-based screen, all four molecules specifically target RIP1 kinase in necroptotic cells. Importantly, we found that all necrostatins lack activity in RIP1-deficient cells. This finding highlights the role of RIP1 kinase as a critical druggable target in necroptosis pathway. Furthermore, selectivity profiling against a panel of human kinases, revealed that necrostatin-1 (Nec-1), displays exquisite selectivity for RIP1. The critical role of RIP1 kinase in necroptosis raises the need to develop methods to study RIP1 kinase activity in vitro to understand the molecular mechanism of RIP1 kinase inhibition by the different necrostatins. For that, we optimized the expression and purification of recombinant RIP1 kinase in Sf9 cells. Using recombinant kinase, we have developed several new assays to measure both catalytic activity and RIP1/necrostatin binding interactions in vitro, including methods utilizing scintillation proximity and homogeneous time resolved fluorescence. In addition, fluorescent and photo-crosslinkable necrostatins analogs were developed to directly access binding of the inhibitors to recombinant RIP1 kinase. These assays provide useful tools to study RIP1 kinase activity and can be adapted to high throughput screening for use in the discovery of novel RIP1 kinase inhibitors. We further discovered that all necrostatins likely target overlapping, but not identical sites in the active center of RIP1 kinase and these differences in binding sites result in significantly different modes of RIP1 inhibition. Strikingly, this translates into dramatic differences in the potency of inhibition of necroptosis in different cell types by the distinct structural classes of necrostatins. Notably, some of these molecules differentiate between human and mouse RIP1 kinases, despite ∼85% identity in protein sequence. Using bioinformatics, molecular modeling and site-directed mutagenesis approaches, we begun to look into critical structural determinants that may be responsible for the activity of necrostatins and their selectivity towards particular species of RIP1 kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY27-03. doi:1538-7445.AM2012-SY27-03

Nat. Med. 14, 1363–1369 (2008); published online 30 November 2008; corrected after print 4 December 2008 In the version of this article initially published, the title included a misspelling—'heterotropic' should have been 'heterotopic'. Additionally, the fourth and fifth sentences of the abstract were incorrectly worded and have been corrected to state more clearly the role of Ad.

The reaction of N-cyano-N′-(6-amino-2-pyridyl)acetamidine (5a) and homophthalic anhydride followed by ring closure of the 2-[2-(carboxymethyl)phenyl]-5-methyl-1,3,4,6,9b-pentaazaphenalene intermediate (4a) gave 5-methyl-13-oxo-13H-4,6,7,13a,13c-pentaazabenzo[hi]chrysene (8a). An analogous series starting with 3-N-(6-amino-2-pyridyl)amino-2-cyano-2-butenenitrile (5b) in place of 5a gave in two steps 5-methyl-13-oxo-13-H-4,7,13a,13c-tetraazabenzo[hi]chrysene-6-carbonitrile (8b). Elemental analysis, ir and pmr spectra of 8a, 8b and several new model compounds aided in confirming the structures of 8a and 8b. The synthesis of one of these model compounds for 5b and phenylacetic anhydride led surprisingly to 2-methyl-9-phenyl-7H-3,7,-10,10b-tetraazacyclohepta[de]naphthalene (10) in addition to the expected 2-benzyl-4-cyano-5-methyl-1,3,-6,9b-tetraazaphenalene (7b).

SummaryMicrotubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest.